The regulation of glucose homeostasis is a complex process, which is disrupted in disease states such as type 2 diabetes. Insulin is the primary hormone regulating glucose homeostasis. Insulin stimulates glucose uptake in muscle and fat by causing the movement of GLUT4 glucose transporters out of intracellular membranes and to the cell surface. This effect of insulin is impaired in the setting of overnutrition, inactivity, ad genetic predisposition, resulting in insulin resistance and contributing to the development of diabetes. Therefore, to understand the pathogenesis of metabolic disease, it is necessary to understand the molecular mechanisms that control the targeting of GLUT4 among intracellular membranes, and by which this targeting is modulated by insulin. Previous work by this project identified the TUG protein as a regulator of GLUT4 targeting and glucose uptake in muscle, as in fat, and showed that this mechanism controls energy expenditure in mice. The data support a model in which GLUT4 is specifically retained within cells not stimulated by insulin by a protein complex containing TUG. Insulin then mobilizes GLUT4, in part by triggering TUG endoproteolytic cleavage. Cleavage may coordinate the regulation of glucose uptake with other effects on physiology and metabolism, which can result from the action of proteins that are produced by cleavage or co-regulated with GLUT4. This project focuses on the cleavage mechanism itself, and on how the ability of insulin to stimulate cleavage may be modulated to control insulin sensitivity. Aim 1 will define the relationship between TUG-bound vesicles and the insulin-responsive GLUT4 storage vesicles, and will study how TUG interacts with vesicle proteins, particularly IRAP, and undergoes proteolytic cleavage. To understand the physiologic role of TUG in the regulation in muscle, we will create muscle-specific TUG knockout mice and study glucose homeostasis in these animals. Aim 2 will study how acetylation of the TUG protein affects insulin sensitivity. We will test the hypothesis that acetylation modulates the interaction of the TUG carboxyl terminus with ACBD3, a protein present at the Golgi matrix, to control the size of an insulin-responsive pool of GLUT4. We will further study if a sirtuin protein regulates this acetylation to control insulin sensitivity in muscle, using knockout mice. We anticipate that, together, these studies will result in an improved understanding of molecular mechanisms regulating glucose metabolism and energy expenditure, with implications for the prediction, prevention, and treatment of diabetes and the metabolic syndrome.